Identification of Peptides in Aggregates Formed during Hydrolysis of β-Lactoglobulin B with a Glu and Asp Specific Microbial Protease

Abstract

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate, enzyme and time of hydrolysis

“…The low ACE-inhibitory activity of the hydrolysates made with BLP (Table 3) is in line with the low affinity of ACE for substrates with C-terminal dicarboxylic acids (Li et al, 2004). Furthermore, peptides formed by the action of BLP may aggregate and precipitate (Otte, Ipsen, Bauer, Bjerrum, & Waninge, 2005;Otte, Lomholt, Halkier, & Qvist, 2000), rendering fewer peptides available for inhibition of the enzyme.…”

Peptide–peptide and protein–peptide interactions in mixtures of whey protein isolate and whey protein isolate hydrolysates

“…2), which was also observed by Otte et al (1997Otte et al ( , 2000. Because in WPI hydrolysates the simultaneous presence of intact b-lg and aggregating peptides was not possible, the interaction between these peptides and whey proteins was studied by titrating the hydrolysates with WPI.…”

“…Aggregates formed during hydrolysis of b-lg with BLP consist of peptides of intermediate size (2-6 kDa) held together by mainly hydrophobic interactions (Otte et al, 1997). The aggregates are made up of six to seven major peptides, of which four have been identified (Otte, Lomholt, Halkier, & Qvist, 2000). Hydrolysis of a-lactalbumin (a-la), the second major protein in bovine whey, with BLP also leads to the formation of aggregates and, under certain conditions, to so-called nanotubules (Graveland-Bikker, Ipsen, Otte, & de Kruif, 2004;Otte, Ipsen, Ladefogrd, & Sorensen, 2004).…”

Fractionation and characterisation for antioxidant activity of hydrolysed whey protein

“…Increased aggregate formation, accompanied by a decrease in free peptide fractions, of hydrolysed WPI and β-lactoglobulin with heat treatment has been reported previously. 18,19 These large polypeptides, also contained in fractions a1 and p1, might include, in addition to native immunoglobulins (IgG) and serum albumin (BSA), adducts of hydrolysed peptides which were hydrophobically associated as a result of heat treatment before and after hydrolysis. The separation of small-MW fractions of W/F was not effectively achieved, as the average MWs of f4, f5 and f6 (4.8, 1.8 and 0.5 kDa respectively) were too small to allow for different partitions in the gel.…”